In this competitive revision of R01GM105993, a new aim is introduced to use camelid variable heavy chain antibody domains (nanobodies) as structural scaffolds and functional probes to investigate the interaction between Ric-8A and Gai1. R01GM105993 funds research to understand how G proteins are activated in the cytoplasm of the cell by a protein factor called Ric-8A. Heterotrimeric G proteins modulate cell metabolism, secretion, electrical conductivity, gene transcription, cell division and cellular motility, and therefore are essential to life in the domain of eukaryotes to which humans belong. Misregulation of G proteins is associated with cancer and a range of other diseases of relevance to general medicine. While most processes controlled by heterotrimeric G proteins occur at cell membranes, recent research has shown that G alpha subunits (Ga) also control certain events in cell cytoplasm. Important among these is asymmetric cell division, which is essential for embryonic development. Ric-8A is critical regulator of Ga in this process. Ric-8A is a Guanine nucleotide Exchange Factor (GEF) that activates Ga by catalyzing the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) at the active site of Ga. The intermediate in this reaction is the nucleotide-free Ga:Ric-8A complex. Describing the structural changes that occur when Ric-8A binds to Ga*GDP is key to understanding how Ric-8A activates Ga. Ric-8A is also a chaperone that promotes proper folding of Ga in cells. The first three aims of R01GM105993 address the structure and the dynamic behavior of the Ric-8A:Gai1 complex, and the structural changes in Ga and Ric-8A that accompany its formation. In these aims, Double Electron-Electron Resonance (DEER) spectroscopy, Hydrogen-Deuterium eXchange coupled with Mass Spectrometry (HDX-MS) and single molecule Frster Resonance Energy Transfer (smFRET) experiments will be conducted to probe changes in Gai1 and Ric-8A structure and dynamics. The new aim is to use nanobodies to stabilize discrete structural states of the Ric-8A:Gai1 complex to render them amenable to crystallization and structure determination by X-ray crystallography. Nanobodies that alter the GEF or chaperone activity of Ric-8A will be used to identify functional sites in Ric-8A and Ga using HDX-MS. DEER and smFRET will be used to determine whether nanobodies that impair or enhance Ric-8A activity either attenuate or amplify global structural changes or protein dynamics.